Proficiency test sample media for single and mixed pure cultures of water pollution indicator bacteria.

Two transport media, NYSDH-1 and NYSDH-2, were developed for use in a split bacteriological water sample program. The media maintained 88% viability of inoculated organisms for at least 48 h, and the samples do not require special handling or reconstitution. Procedures for preparing and shipping the samples to participating laboratories were developed. A reference set of samples was analyzed in laboratories certified by either New York State or the Environmental Protection Agency. A statistical analysis was performed, and the results indicate that the media are suitable for integration into a laboratory quality control program.     

Proficiency test sample media for single and mixed pure cultures of water pollution indicator bacteria

Two transport media, NYSDH-1 and NYSDH-2, were developed for use in a split bacteriological water sample program. The media maintained 88% viability of inoculated organisms for at least 48 h, and the samples do not require special handling or reconstitution. Procedures for preparing and shipping the samples to participating laboratories were developed. A reference set of samples was analyzed in laboratories certified by either New York State or the Environmental Protection Agency. A statistical analysis was performed, and the results indicate that the media are suitable for integration into a laboratory quality control program.     

Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials.     

水产品微生物实验室质量控制探讨

从检验人员、仪器设备、培养基与试剂、采样、样品接收与预处理、检验质量控制、参考菌株及保存、检验报告与结果质量等方面,对水产品微生物学检验质量控制进行了探讨,以期更好地开展水产品微生物检验,提高水产品卫生质量,保障消费者饮食安全。     

Numerical Diagnostic Key for the Identification of Enterobacteriaceae

A numerical diagnostic key for enteric organisms is described which permits the identification of typical strains and of biochemical variants with high accuracy. Unknown strains are inoculated into a basic set of five media which permit the testing of eight biochemical reactions. The positive reactions are assigned points, and the score of a strain is added up, after which the identification of the strain is obtained from a table. In many instances, the final identification is obtained with this set of biochemical tests; and, in other instances, a small number of additional tests are required to distinguish between organisms giving the same score in the basic set of biochemical tests. The key permits an accurate, rapid, and economical differentiation of the typical and the more common atypical biotypes of enteric organisms in the clinical laboratory.     

Laboratory Identification of Aeromonads from Man and Other Animals

Forty-eight isolates of aeromonads from clinical specimens were studied. The laboratory procedures employed and the results obtained are reported in the hope that they may be of assistance to other workers in the field of diagnostic microbiology. Capability of identifying these organisms in the laboratory will contribute to a better understanding of the role of this group of organisms in human disease as well as in diseases in other animals.     

Identification of the Mima-Herellea Group of Organisms and Selected Members of the Genus Neisseria

To determine a rapid and reliable protocol for the differentiation of Mima polymorpha, M. polymorpha var. oxidans, Herellea, Bacterium antitratum, Neisseria gonorrheae, and other related members of the genus Neisseria, reference cultures were examined on a variety of microbiological media. The media were selected because of the typical morphological and biochemical characteristics exhibited by the test organisms and were those media which would be readily available in a microbiology facility. After compiling data obtained from 21,714 observations, an inclusive protocol is presented which has proven to be quite adequate for the identification of these particular gram-negative bacteria.     

Extraction of total nucleic acids from bacterial isolates using the bioMérieux NucliSENS easyMAG total nucleic acid extractor

The BioMerieux NucliSENS easyMAG total nucleic acid extractor was evaluated for use on bacterial isolates in the clinical microbiology laboratory. Forty eight isolates were extracted, yielding quantifiable amounts of DNA for all isolates. The easyMAG is appropriate for DNA extraction from bacterial isolates and will be incorporated in the clinical laboratory.     

Development of rapid phenotypic system for the identification of Gram-negative oxidase-positive bacilli in resource-limited settings

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology. The diagnosis and surveillance of diseases is dependent, to a great extent, on laboratory services, which cannot function without effective reliable reagents and diagnostics. Despite the advancement in microbiology diagnosis globally, resource-limited countries still struggle to provide an acceptable diagnosis quality which helps in clinical disease management and improve their mortality and morbidity data. During this study an indigenous product, Quick Test Strip (QTS) NE, was developed for the rapid identification of biochemically slower group of Gram-negative oxidase-positive bacilli that covers 19 different bacterial genera. Some of the members belonging to these groups are well-established human pathogens, e.g. various species of Vibrio, Pseudomonas, Burkholderia, Aeromonas, Achromobacter and Stenotrophomonas. This study also evaluates the performance of QTS-NE by comparing with genotypic characterization methods. A total of 232 clinical and reference bacterial isolates were tested by three different methods. QTS-NE provides 100% concordant results with other rapid identification and molecular characterization methods and confirms the potential to be used in clinical diagnosis.     

Diagnostic virology laboratory within a microbiology setting.

The virology section at St. Francis Hospital and Medical Center, Connecticut, is not a separate laboratory division but is a part of the microbiology division and is supervised by the same personnel who supervise bacteriology, mycology, mycobacteriology, and serology. Current volume is over 1,000 cultures yearly with 12 to 24 percent positive. Isolates are confirmed and typed by the Connecticut State Health Department Laboratory. Specimen distribution, percentage positive specimens, and distribution of viral isolates are similar to those reported from microbiology laboratories with separate virology laboratories directed by a full-time doctoral-level virologist. Our seven years'' experience demonstrates that a microbiology laboratory without a full-time doctoral-level virologist can provide clinically useful virologic information.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

The Quality of Sputum Smear Microscopy in Public-Private Mix Directly Observed Treatment Laboratories in West Amhara Region,Ethiopia

Ethiopia adopted Public-Private Mix Directly Observed Treatment Short Course Chemotherapy (PPM-DOTS) strategy for tuberculosis (TB) control program. Quality of sputum smear microscopy has paramount importance for tuberculosis control program in resource-poor countries like Ethiopia. A cross-sectional study was conducted to assess the quality of sputum smear microscopy in 37 Public-Private Mix laboratories in West Amhara, Ethiopia. The three external quality assessment methods (onsite evaluation, panel testing and blind rechecking) were employed. Onsite assessment revealed that 67.6% of PPM-DOTS laboratories were below the standard physical space (5 X 6) m². The average monthly workload per laboratory technician was 19.5 (SD±2.9) slides with 12.8% positivity rate. The quality of Acid Fast Bacilli (AFB) staining reagents was sub-standard. The overall agreement for blind rechecking of 1,123 AFB slides was 99.4% (Kappa = 0.97). Reading of 370 AFB panel slides showed 3.5% false reading (Kappa = 0.92). Moreover, the consistency of reading scanty bacilli slides was lower (93%) compared to 1+, 2+ and 3+ bacilli. Based on blind rechecking and panel testing results, PPM-DOTS site laboratories showed good agreement with the reference laboratory. Physical space and qualities of AFB reagents would be areas of intervention to sustain the quality of sputum smear microscopy. Therefore, regular external quality assessment and provision of basic laboratory supplies for TB diagnosis would be the way forward to improve the quality of sputum smear microscopy services in PPM-DOTS laboratories.     

A simple and fast method for determining colony forming units

Aims: To develop a flexible and fast colony forming unit quantification method that can be operated in a standard microbiology laboratory. Methods and Results: A miniaturized plating method is reported where droplets of bacterial cultures are spotted on agar plates. Subsequently, minicolony spots are imaged with a digital camera and quantified using a dedicated plug‐in developed for the freeware program Image J. A comparison between conventional and minicolony plating of industrial micro‐organisms including lactic acid bacteria, Eschericha coli and Saccharomyces cerevisiae showed that there was no significant difference in the results obtained with the methods. Conclusions: The presented method allows downscaling of plating by 100‐fold, is flexible, easy‐to‐use and is more labour‐efficient and cost‐efficient than conventional plating methods. Significance and Impact of the Study: The method can be used for rapid assessment of viable counts of micro‐organisms similar to conventional plating using standard laboratory equipment. It is faster and cheaper than conventional plating methods.     

Growth characteristics of microorganisms on commercially available animal-free alternatives to tryptic soy medium

The growth characteristics of a range of bacteria and fungi that are routinely used in quality control practices were compared for two representative vegetable-based tryptic soy formulations. All of the representative microorganisms grew well on the vegetable-based media and the media provided suitable recoveries of the organisms following simulated storage. Subtle phenotypic changes were observed between cultures grown on different media, but these did not significantly change the strain identification.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

Microarray-based genetic identification of beneficial organisms as a new tool for quality control of laboratory cultures

The use of beneficial organisms to help control pests and pathogens in field and greenhouse crops is constantly increasing. Insects and mites are commonly used as beneficial organisms and, nowadays, rearing companies have to produce them in large quantities. Because of the peculiarities of laboratory culture conditions, the quality of lab-reared organisms generally degrades over time. To maintain high fitness levels, cultures are refreshed with field specimens at regular intervals. However, this bears the risk of contaminating laboratory cultures with species or strains other than the intended natural enemy. To ensure that the correct species is produced and also to facilitate surveys after field release, we have developed a diagnostic microarray for identification of beneficial species. Probes have been designed from the different haplotypes of a fragment of the mitochondrial cytochrome oxidase I (COI) gene of each species. Hybridization of labeled PCR amplicons of COI on the microarray chip allows precise identification of 28 economically relevant arthropod species.     

Characterization of the bacterial microflora of the tympanic cavity of eastern box turtles with and without aural abscesses

Aerobic bacterial cultures of the tympanic cavity of the middle ear were performed in eight eastern box turtles (Terrapene carolina carolina) with aural abscesses and 15 eastern box turtles without aural abscesses (controls) that were admitted to The Wildlife Center of Virginia, Virginia, USA during 2003. Twenty-two bacterial isolates were identified from 17 turtles including 10 gram-negative and 12 gram-positive bacteria. Ten of 15 control animals had bacterial growth, resulting in identification of 13 bacteria, including six gram-negative and seven gram-positive agents. Seven of eight turtles with aural abscesses had bacterial growth, and 10 isolates were identified, including four gram-negative and six gram-positive organisms. The most frequently isolated bacteria from control animals were Micrococcus luteus (n = 3) and Pantoea agglomerans (n = 2). Morganella morganii (n = 2) was the only species isolated from the tympanic cavity of more than one turtle with aural abscesses. Staphylococcus epidermidis (n = 2) was the only species isolated from both groups. A trend toward greater bacterial growth in tympanic cavities of affected turtles compared with turtles without aural abscesses was noted. No single bacterial agent was responsible for aural abscesses in free-ranging eastern box turtles in this study, an observation consistent with the hypothesis that aerobic bacteria are not primary pathogens, but secondary opportunistic invaders of environmental origin.     

The application of chromogenic media in clinical microbiology

Since 1990, a wide range of chromogenic culture media has been made commercially available providing useful tools for diagnostic clinical microbiology. By the inclusion of chromogenic enzyme substrates targeting microbial enzymes, such media are able to target pathogens with high specificity. Examples of target pathogens include Staphylococcus aureus, Streptococcus agalactiae, Salmonella spp. and Candida spp. The inclusion of multiple chromogenic substrates into culture media facilitates the differentiation of polymicrobial cultures, thus allowing for the development of improved media for diagnosis of urinary tract infections and media for the enhanced discrimination of yeasts. The purpose of this review is to provide some insight into how such media work and appraise their utility in routine clinical diagnostics, in comparison with conventional media.     

Synthesis of M Protein by Group A Hemolytic Streptococci in Completely Synthetic Media During Steady-State Growth

Strains of type 6 (S 43) and type 14 group A streptococci were grown with M-protein production in the presence of chemically defined synthetic media slightly modified from that previously employed for the growth of a nonproducer of M protein (type 4). The M protein, which is associated with virulence in group A streptococcus, was previously produced in growing cultures only with complex media. The bacterial growth with the biosynthesis of M protein in synthetic medium was obtained by successive adaptation in steady-state culture with decreasing amounts of Todd-Hewitt broth. The synthesis continued for at least 480 generations at pH 7.3 and with a generation time of 84 min. Glucose was the limiting nutrilite and the concentration of reducing agents in the medium was critical. The M protein was identified by gel diffusion against type-specific antisera from the Communicable Disease Center and from R. Lancefield. The yield of M protein obtained from organisms grown in the continuous-culture device was comparable to that from standard broth stationary cultures.